The present invention is generally directed to a method for producing industrially relevant quantities of catalytically enhanced recombinant enzymes. More particularly, the present invention is generally directed to such a process wherein the gene encoding the enzyme is a mutant that enables a catalytic cysteine or serine to be substituted with the selenium analog, i.e. selenocysteine.
Industrially relevant production of selenoproteins in non-animal or single celled systems has proven challenging because eukaryotic and bacterial selenocysteine insertion sequences (SECIS) operate differently. Bacterial SECISs are usually located immediately downstream of the UGA codon encoding selenocysteine. Thus, bacterial SECISs are translated. In contrast, typical eukaryotic SECISs are part of the 3′ untranslated region (UTR) of the mRNA. The upshot of this architectural difference between bacterial and eukaryotic SECISs is that in order to express a eukaryotic gene in a bacterial cell you must add codons to the gene, which results in adding amino acids to the gene product. Accordingly, expressing a eukaryotic gene in bacteria is certain to affect the structure and likely to deleteriously affect the function of the recombinant protein.
Previously, others have demonstrated that Chlamydomonas has a eukaryotic SECIS. This suggested that it is possible to produce selenocysteine proteins in Chlamydomonas. However, the prior work was silent as to the catalytic benefit conferred by artificially replacing wild-type cysteine with selenocysteine and methods for secreting recombinant selenocysteine-containing proteins from Chlamydomonas under the control of a tightly regulated gene promoter.
The present invention provides methods for replacing cysteine and/or serine with selenocysteine. More particularly, the present invention provides a method of producing recombinant selenocysteine-containing proteins using any gene source, prokaryotic or eukaryotic, wherein the protein is catalytically enhanced through replacing cysteine and/or serine with selenocysteine. Accordingly, the present invention is novel and nonobvious, and thus deserves broad patent protection.